The fusion of neurotransmitter-containing (synaptic) vesicles is the primary signaling event between neurons. Because of this, identifying how synaptic vesicle fusion is produced and regulated is 1 of the central goals of neurobiology. Although most of the proteins associated with synaptic vesicles have been identified, their concentration in vesicles and whether concentration or isoform makeup varies among vesicles from different types of synapses or under different conditions is not known. To address these questions, we propose to develop and utilize high-resolution imaging and single-molecule detection techniques that will allow quantification of proteins in single synaptic vesicles. We will also develop a nanofluidic sorting technique that will allow us to analyze different subpopulations of vesicles. This work brings the development of novel technological tools to bear on fundamental questions in neurobiology. It will lay the foundation for the development of therapies aimed at modifying the action of subpopulations of synapses. In addition, the nanofluidic sorting technique we propose to develop also represents a major advancement in subcellular fractionation and should find wide spread applications in cell biology and neuroscience. Our specific aims are: 1. To quantify the number of each class of synaptic vesicle protein in synaptic vesicles using high-resolution imaging and high-sensitivity detection techniques that allows the counting of single fluorescent antibody molecules. 2. To test the hypothesis that synaptic vesicle protein number is regulated, by quantifying proteins in vesicles isolated from animals heterozygous for synaptic vesicle protein gene deletions. 3. To develop a nanoscale fluorescence activated vesicle sorting (nFAVS) technique to isolate and analyze vesicles containing different neurotransmitters and vesicles from different regions of the CNS. 4. To examine the effects of aberrant synaptic activity on vesicular protein composition.